ABSTRACT This proposal is being submitted as part of a group of linked merit review applications from nationwide VA experts in colorectal cancer (CRC) who have formed a colorectal cancer cell-genomics consortium (VA4C) after participating in a successful field-based meeting in Chicago in May 2017 (funded by VA ORD). Specifically, this proposal will be part of a cluster of merit review projects that are aimed at advancing our understanding of CRC pathogenesis and treatment response with a focus on the roles of the microbiome and CRC stem cells. The importance of the intestinal microbiome to the development and progression of CRC is supported by a range of evidence including epidemiological links between diet and CRC risk, reproducible associations of specific microbes such as Fusobacterium with human CRC, mouse models indicating augmentation of CRC formation and metastasis by microbiota, and mouse models showing that the microbiome modulates chemotherapy response. We hypothesize that: 1) The intestinal microbiome, through direct invasion of tumor tissue and release of bioactive microbial products, is associated with specific CRC clinical/histologic phenotypes and molecular subclasses defined by transcriptional profiles. 2) The intestinal microbiome influences the risk of recurrence after surgery and chemotherapy by modulating anti-tumor immunity and the metabolism of chemotherapeutics. 3) The treatment of locally advanced CRC with surgical resection and chemotherapy alters the intestinal microbiome, which may influence the likelihood of future recurrences. To study these hypotheses, in Aim 1 we will utilize the multi-center clinical infrastructure of this CMA to collect fecal and tumor samples from 400 male and female Veterans with stage I-III CRC for analysis of the microbiome, metabolome, and transcriptome. Intestinal microbes and metabolites will be identified that are associated with tumor phenotype and molecular signature, as defined by gene transcripts, functional pathways and non-coding RNAs. In Aim 2, we will perform longitudinal follow-up of 200 Veterans with stage II- III colorectal cancer to elucidate baseline microbes and metabolites that predict subsequent risk for recurrence. In addition, we will obtain fecal samples before, during and after surgery then chemotherapy, and annually thereafter to identify microbial shifts with treatment and their association with future recurrence. In Aim 3, we will link compositional and functional shifts in the intestinal microbiome to CRC by using the C57BL/6 ApcMin strain of mice that is highly susceptible to spontaneous intestinal adenoma formation. We will examine the effect of colonization with human microbiota obtained from patients with high or low immune infiltration and immune transcriptional state, to assess adenoma/CRC formation and the intratumor immune response. We will also study the response to chemotherapy in mice engrafted with CRC cells and colonized with the stool of patients that had a recurrence, or a sustained remission, to evaluate microbiome-mediated differences in therapeutic response.